Incerto-thalamic modulation of fear via GABA and dopamine
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology
Venkataraman, A;Hunter, SC;Dhinojwala, M;Ghebrezadik, D;Guo, J;Inoue, K;Young, LJ;Dias, BG;
PMID: 33864008 | DOI: 10.1038/s41386-021-01006-5
Fear generalization and deficits in extinction learning are debilitating dimensions of Post-Traumatic Stress Disorder (PTSD). Most understanding of the neurobiology underlying these dimensions comes from studies of cortical and limbic brain regions. While thalamic and subthalamic regions have been implicated in modulating fear, the potential for incerto-thalamic pathways to suppress fear generalization and rescue deficits in extinction recall remains unexplored. We first used patch-clamp electrophysiology to examine functional connections between the subthalamic zona incerta and thalamic reuniens (RE). Optogenetic stimulation of GABAergic ZI → RE cell terminals in vitro induced inhibitory post-synaptic currents (IPSCs) in the RE. We then combined high-intensity discriminative auditory fear conditioning with cell-type-specific and projection-specific optogenetics in mice to assess functional roles of GABAergic ZI → RE cell projections in modulating fear generalization and extinction recall. In addition, we used a similar approach to test the possibility of fear generalization and extinction recall being modulated by a smaller subset of GABAergic ZI → RE cells, the A13 dopaminergic cell population. Optogenetic stimulation of GABAergic ZI → RE cell terminals attenuated fear generalization and enhanced extinction recall. In contrast, optogenetic stimulation of dopaminergic ZI → RE cell terminals had no effect on fear generalization but enhanced extinction recall in a dopamine receptor D1-dependent manner. Our findings shed new light on the neuroanatomy and neurochemistry of ZI-located cells that contribute to adaptive fear by increasing the precision and extinction of learned associations. In so doing, these data reveal novel neuroanatomical substrates that could be therapeutically targeted for treatment of PTSD.
McKinnon C, De Snoo ML, Gondard E, Neudorfer C, Chau H, Ngana SG, O'Hara DM, Brotchie JM, Koprich JB, Lozano AM, Kalia LV, Kalia SK
PMID: 32059750 | DOI: 10.1186/s40478-020-0894-0
Parkinson's disease is a progressive neurodegenerative disorder characterised by the accumulation of misfolded ?-synuclein in selected brain regions, including the substantia nigra pars compacta (SNpc), where marked loss of dopaminergic neurons is also observed. Yet, the relationship between misfolded ?-synuclein and neurotoxicity currently remains unclear. As the principal route for degradation of misfolded proteins in mammalian cells, the ubiquitin-proteasome system (UPS) is critical for maintenance of cellular proteostasis. Misfolded ?-synuclein impairs UPS function and contributes to neuronal death in vitro. Here, we examine its effects in vivo using adeno-associated viruses to co-express A53T ?-synuclein and the ubiquitinated reporter protein UbG76V-GFP in rat SNpc. We found that ?-synuclein over-expression leads to early-onset catalytic impairment of the 26S proteasome with associated UPS dysfunction, preceding the onset of behavioural deficits and dopaminergic neurodegeneration. UPS failure in dopaminergic neurons was also associated with selective accumulation of ?-synuclein phosphorylated at the serine 129 residue, which has previously been linked to increased neurotoxicity. Our study highlights a role for ?-synuclein in disturbing proteostasis which may contribute to neurodegeneration in vivo
Liu, Z;Yang, N;Dong, J;Tian, W;Chang, L;Ma, J;Guo, J;Tan, J;Dong, A;He, K;Zhou, J;Cinar, R;Wu, J;Salinas, AG;Sun, L;Kumar, M;Sullivan, BT;Oldham, BB;Pitz, V;Makarious, MB;Ding, J;Kung, J;Xie, C;Hawes, SL;Wang, L;Wang, T;Chan, P;Zhang, Z;Le, W;Chen, S;Lovinger, DM;Blauwendraat, C;Singleton, AB;Cui, G;Li, Y;Cai, H;Tang, B;
PMID: 35715418 | DOI: 10.1038/s41467-022-31168-9
Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase β (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.
Wimalasena, NK;Taub, DG;Shim, J;Hakim, S;Kawaguchi, R;Chen, L;El-Rifai, M;Geschwind, D;Dib-Hajj, SD;Waxman, SG;Woolf, CJ;
PMID: 37003485 | DOI: 10.1016/j.expneurol.2023.114393
Gain-of-function mutations in Scn9a, which encodes the peripheral sensory neuron-enriched voltage-gated sodium channel Nav1.7, cause paroxysmal extreme pain disorder (PEPD), inherited erythromelalgia (IEM), and small fiber neuropathy (SFN). Conversely, loss-of-function mutations in the gene are linked to congenital insensitivity to pain (CIP). These mutations are evidence for a link between altered sodium conductance and neuronal excitability leading to somatosensory aberrations, pain, or its loss. Our previous work in young adult mice with the Nav1.7 gain-of-function mutation, I228M, showed the expected DRG neuron hyperexcitability, but unexpectedly the mice had normal mechanical and thermal behavioral sensitivity. We now show that with aging both male and female mice with this mutation unexpectedly develop a profound insensitivity to noxious heat and cold, as well skin lesions that span the body. Electrophysiology demonstrates that, in contrast to young mice, aged I228M mouse DRGs have a profound loss of sodium conductance and changes in activation and slow inactivation dynamics, representing a loss-of-function. Through RNA sequencing we explored how these age-related changes may produce the phenotypic changes and found a striking and specific decrease in C-low threshold mechanoreceptor- (cLTMR) associated gene expression, suggesting a potential contribution of this DRG neuron subtype to Nav1.7 dysfunction phenotypes. A GOF mutation in a voltage-gated channel can therefore produce over a prolonged time, highly complex and unexpected alterations in the nervous system beyond excitability changes.
Wadsworth, HA;Anderson, EQ;Williams, BM;Ronström, JW;Moen, JK;Lee, AM;McIntosh, JM;Wu, J;Yorgason, JT;Steffensen, SC;
PMID: 36802012 | DOI: 10.1007/s12035-023-03263-5
The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system, consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc), underlies the reward properties of ethanol (EtOH) and nicotine (NIC). We have shown previously that EtOH and NIC modulation of DA release in the NAc is mediated by α6-containing nicotinic acetylcholine receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and that α6*-nAChRs may be a molecular target for low-dose EtOH. However, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on GABAergic modulation of VTA GABA neurons and VTA GABAergic input to cholinergic interneurons (CINs) in the NAc. Low-dose EtOH enhanced GABAergic input to VTA GABA neurons that was blocked by knockdown of α6*-nAChRs. Knockdown was achieved either by α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice or by superfusion of the α-conotoxin MII[H9A;L15A] (MII). Superfusion of MII blocked EtOH inhibition of mIPSCs in NAc CINs. Concomitantly, EtOH enhanced CIN firing rate, which was blocked by knockdown of α6*-nAChRs with α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice. The firing rate of CINs was not enhanced by EtOH in EtOH-dependent mice, and low-frequency stimulation (LFS; 1 Hz, 240 pulses) caused inhibitory long-term depression at this synapse (VTA-NAc CIN-iLTD) which was blocked by knockdown of α6*-nAChR and MII. Ethanol inhibition of CIN-mediated evoked DA release in the NAc was blocked by MII. Taken together, these findings suggest that α6*-nAChRs in the VTA-NAc pathway are sensitive to low-dose EtOH and play a role in plasticity associated with chronic EtOH.
Gervasi NM, Scott SS, Aschrafi A, Gale J, Vohra SN, MacGibeny MA, Kar AN, Gioio AE, Kaplan BB.
PMID: 27095027 | DOI: 10.1261/rna.053272.115.
Synthesis and regulation of catecholamine neurotransmitters in the central nervous system are implicated in the pathogenesis of a number of neuropsychiatric disorders. To identify factors that regulate the presynaptic synthesis of catecholamines, we tested the hypothesis that the rate-limiting enzyme of the catecholamine biosynthetic pathway, tyrosine hydroxylase (TH), is locally synthesized in axons and presynaptic nerve terminals of noradrenergic neurons. To isolate pure axonal mRNA and protein, rat superior cervical ganglion sympathetic neurons were cultured in compartmentalized Campenot chambers. qRT-PCR and RNA in situ hybridization analyses showed that TH mRNA is present in distal axons. Colocalization experiments with nerve terminal marker proteins suggested that both TH mRNA and protein localize in regions of the axon that resemble nerve terminals (i.e., synaptic boutons). Analysis of polysome-bound RNA showed that TH mRNA is present in polysomes isolated from distal axons. Metabolic labeling of axonally synthesized proteins labeled with the methionine analog, L-azidohomoalanine, showed that TH is locally synthesized in axons. Moreover, the local transfection and translation of exogenous TH mRNA into distal axons facilitated axonal dopamine synthesis. Finally, using chimeric td-Tomato-tagged constructs, we identified a sequence element within the TH 3'UTR that is required for the axonal localization of the reporter mRNA. Taken together, our results provide the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated. These findings raise the interesting possibility that the biosynthesis of the catecholamine neurotransmitters is locally regulated in the axon and/or presynaptic nerve terminal.
Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology
Velazquez-Sanchez, C;Muresan, L;Marti-Prats, L;Belin, D;
PMID: 36635597 | DOI: 10.1038/s41386-022-01522-y
Some compulsive disorders have been considered to stem from the loss of control over coping strategies, such as displacement. However, the cellular mechanisms involved in the acquisition of coping behaviours and their subsequent compulsive manifestation in vulnerable individuals have not been elucidated. Considering the role of the locus coeruleus (LC) noradrenaline-dependent system in stress and related excessive behaviours, we hypothesised that neuroplastic changes in the LC may be associated with the acquisition of an adjunctive polydipsic water drinking, a prototypical displacement behaviour, and the ensuing development of compulsion in vulnerable individuals. Thus, male Sprague Dawley rats were characterised for their tendency, or not, to develop compulsive polydipsic drinking in a schedule-induced polydipsia (SIP) procedure before their fresh brains were harvested. A new quantification tool for RNAscope assays revealed that the development of compulsive adjunctive behaviour was associated with a low mRNA copy number of the plasticity marker Arc in the LC which appeared to be driven by specific adaptations in an ensemble of tyrosine hydroxylase (TH)+, zif268- neurons. This ensemble was specifically engaged by the expression of compulsive adjunctive behaviour, not by stress, because its functional recruitment was not observed in individuals that no longer had access to the water bottle before sacrifice, while it consistently correlated with the levels of polydipsic water drinking only when it had become compulsive. Together these findings suggest that downregulation of Arc mRNA levels in a population of a TH+/zif268- LC neurons represents a signature of the tendency to develop compulsive coping behaviours.
Gu, X;Zhang, YZ;O'Malley, JJ;De Preter, CC;Penzo, M;Hoon, MA;
PMID: 36894654 | DOI: 10.1038/s41593-023-01268-w
Supraspinal brain regions modify nociceptive signals in response to various stressors including stimuli that elevate pain thresholds. The medulla oblongata has previously been implicated in this type of pain control, but the neurons and molecular circuits involved have remained elusive. Here we identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. Upon activation, these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord. This pathway is sufficient to attenuate injury-induced heat allodynia and is required for counter-stimulus induced analgesia to noxious heat. Our findings define a component of the pain modulatory system that regulates nociceptive responses.
Proc Natl Acad Sci U S A.
Shen H, Marino RAM, McDevitt RA, Bi GH, Chen K, Madeo G, Lee PT, Liang Y, De Biase LM, Su TP, Xi ZX, Bonci A.
PMID: 30442663 | DOI: 10.1073/pnas.1800886115
A subset of midbrain dopamine (DA) neurons express vesicular glutamate transporter 2 (VgluT2), which facilitates synaptic vesicle loading of glutamate. Recent studies indicate that such expression can modulate DA-dependent reward behaviors, but little is known about functional consequences of DA neuron VgluT2 expression in neurodegenerative diseases like Parkinson's disease (PD). Here, we report that selective deletion of VgluT2 in DA neurons in conditional VgluT2-KO (VgluT2-cKO) mice abolished glutamate release from DA neurons, reduced their expression of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB), and exacerbated the pathological effects of exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Furthermore, viral rescue of VgluT2 expression in DA neurons of VglutT2-cKO mice restored BDNF/TrkB expression and attenuated MPTP-induced DA neuron loss and locomotor impairment. Together, these findings indicate that VgluT2 expression in DA neurons is neuroprotective. Genetic or environmental factors causing reduced expression or function of VgluT2 in DA neurons may place some individuals at increased risk for DA neuron degeneration. Therefore, maintaining physiological expression and function of VgluT2 in DA neurons may represent a valid molecular target for the development of preventive therapeutic interventions for PD.
Noncanonical Genomic Imprinting Effects in Offspring
Bonthuis PJ, Huang WC, Stacher Hörndli CN, Ferris E, Cheng T, Gregg C.
Here, we describe an RNA-sequencing (RNA-seq)-based approach that accurately detects even modest maternal or paternal allele expression biases at the tissue level, which we call noncanonical genomic imprinting effects. We profile imprinting in the arcuate nucleus (ARN) and dorsal raphe nucleus of the female mouse brain as well as skeletal muscle (mesodermal) and liver (endodermal). Our study uncovers hundreds of noncanonical autosomal and X-linked imprinting effects. Noncanonical imprinting is highly tissue-specific and enriched in the ARN, but rare in the liver. These effects are reproducible across different genetic backgrounds and associated with allele-specific chromatin. Using in situ hybridization for nascent RNAs, we discover that autosomal noncanonical imprinted genes with a tissue-level allele bias exhibit allele-specific expression effects in subpopulations of neurons in the brain in vivo. We define noncanonical imprinted genes that regulate monoamine signaling and determine that these effects influence the impact of inherited mutations on offspring behavior.
Steinkellner T, Zell V, Farino ZJ, Sonders MS, Villeneuve M, Freyberg RJ, Przedborski S, Lu W, Freyberg Z, Hnasko TS.
PMID: 29337309 | DOI: 10.1172/JCI95795
Parkinson's disease is characterized by the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). DA neurons in the ventral tegmental area are more resistant to this degeneration than those in the SNc, though the mechanisms for selective resistance or vulnerability remain poorly understood. A key to elucidating these processes may lie within the subset of DA neurons that corelease glutamate and express the vesicular glutamate transporter VGLUT2. Here, we addressed the potential relationship between VGLUT expression and DA neuronal vulnerability by overexpressing VGLUT in DA neurons of flies and mice. In Drosophila, VGLUT overexpression led to loss of select DA neuron populations. Similarly, expression of VGLUT2 specifically in murine SNc DA neurons led to neuronal loss and Parkinsonian behaviors. Other neuronal cell types showed no such sensitivity, suggesting that DA neurons are distinctively vulnerable to VGLUT2 expression. Additionally, most DA neurons expressed VGLUT2 during development, and coexpression of VGLUT2 with DA markers increased following injury in the adult. Finally, conditional deletion of VGLUT2 made DA neurons more susceptible to Parkinsonian neurotoxins. These data suggest that the balance of VGLUT2 expression is a crucial determinant of DA neuron survival. Ultimately, manipulation of this VGLUT2-dependent process may represent an avenue for therapeutic development.
Kelberman, M;Rorabaugh, J;Anderson, C;Marriott, A;DePuy, S;Rasmussen, K;McCann, K;Weiss, J;Weinshenker, D;
| DOI: 10.1016/j.neurobiolaging.2023.01.016
Hyperphosphorylated tau in the locus coeruleus (LC) is ubiquitous in prodromal Alzheimer's disease (AD), and LC neurons degenerate as AD progresses. Hyperphosphorylated tau alters firing rates in other brain regions, but its effects on LC neurons are unknown. We assessed single unit LC activity in anesthetized wild-type (WT) and TgF344-AD rats at 6 months, which represents a prodromal stage when LC neurons are the only cells containing hyperphosphorylated tau in TgF344-AD animals, and at 15 months when β-amyloid (Aβ) and tau pathology are both abundant in the forebrain. At baseline, LC neurons from TgF344-AD rats were hypoactive at both ages compared to WT littermates but showed elevated spontaneous bursting properties. Differences in footshock-evoked LC firing depended on age, with 6-month TgF344-AD rats demonstrating aspects of hyperactivity, and 15-month transgenic rats showing hypoactivity. Early LC hyperactivity is consistent with appearance of prodromal neuropsychiatric symptoms and is followed by LC hypoactivity which contributes to cognitive impairment. These results support further investigation into disease stage-dependent noradrenergic interventions for AD.
